Methods of identifying anti-viral agents

ABSTRACT

The present invention provides methods of identifying candidate anti-viral agents.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 60/814,229, filed Jun. 16, 2006, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contact F32A150296awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND

The error-prone replication of RNA viral genomes makes them notoriousfor their ability to evolve resistance to selective agents rapidly andeffectively. For cytoplasmic positive-strand RNA viruses such aspoliovirus, other picornaviruses such as foot-and-mouth disease virus,and more distantly related flaviviruses such as Dengue and West Nileviruses, an infection started by a single genome can quickly becomeheterogeneous, even in the first infected cell. Therefore, a progenygenome containing a newly generated mutation that could confer aselective advantage must replicate and package in the context of anessentially polyploid infection in order to propagate.

There is a need in the art for anti-viral agents that, whenadministered, do not give rise to drug-resistant virus in the earlystages of viral infection.

Literature

Herskowitz (1987) Nature 329:219-222; Crowder and Kirkegaard (2005 July)Nat. Genet. 37(7):701-9 (Epub 2005 Jun. 19).

SUMMARY OF THE INVENTION

The present invention provides methods of identifying candidateanti-viral agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D depict a dominant inhibitor screen for capsid-coding genomeregions.

FIGS. 2A-C depict the effect of mutations in 3D polymerase on the yieldof wild-type virus during co-transfection.

FIGS. 3A-C depict a dominant inhibitor screen in the CRE and 3B-codingregions.

FIGS. 4A-E depict a dominant inhibitor screen for 2A proteinase andVP1-2A cleavage site mutant alleles.

FIGS. 5A-F depict RNA replication or translation requirements fordominance of mutant poliovirus alleles.

FIGS. 6A-C depict superinfections of wild-type and temperature-sensitivepolioviruses.

FIGS. 7A-E depict co-infections of drug-sensitive and drug-resistantviruses.

FIG. 8 is a schematic depiction of an exemplary assay.

FIGS. 9-19 provide amino acid sequences of poliovirus type 1 (Mahoney)proteins VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, 3D, and VP4,respectively.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric forms of nucleotides of any length, eitherribonucleotides or deoxynucleotides. Thus, this term includes, but isnot limited to, single-, double-, or multi-stranded DNA or RNA, genomicDNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The terms “peptide,” “oligopeptide,” “polypeptide,” “polyprotein,” and“protein” are used interchangeably herein, and refer to a polymeric formof amino acids of any length, which can include coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones.

“Recombinant,” as used herein, means that a particular DNA sequence isthe product of various combinations of cloning, restriction, and/orligation steps resulting in a construct having a structural codingsequence distinguishable from homologous sequences found in naturalsystems. Generally, DNA sequences encoding the structural codingsequence can be assembled from cDNA fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene which is capable of being expressed in a recombinanttranscriptional unit. Such sequences can be provided in the form of anopen reading frame uninterrupted by internal nontranslated sequences, orintrons, which are typically present in eukaryotic genes. Genomic DNAcomprising the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5′ or 3′ from the open reading frame,where such sequences do not interfere with manipulation or expression ofthe coding regions. Thus, e.g., the term “recombinant” polynucleotide ornucleic acid refers to one which is not naturally occurring, or is madeby the artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by eitherchemical synthesis means, or by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques. Suchis usually done to replace a codon with a redundant codon encoding thesame or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate adesired combination of functions.

By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

Similarly, a “recombinant polypeptide” or “recombinant polyprotein”refers to a polypeptide or polyprotein which is not naturally occurring,or is made by the artificial combination of two otherwise separatedsegments of amino acid sequences. This artificial combination may beaccomplished by standard techniques of recombinant DNA technology, suchas described above, i.e., a recombinant polypeptide or recombinantpolyprotein may be encoded by a recombinant polynucleotide. Thus, arecombinant polypeptide or recombinant polyprotein is an amino acidsequence encoded by all or a portion of a recombinant polynucleotide.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anRNA virus” includes a plurality of such viruses and reference to “theanti-viral agent” includes reference to one or more anti-viral agentsand equivalents thereof known to those skilled in the art, and so forth.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides methods of identifying candidateanti-viral agents. The invention is based at least on part on thediscovery that choices of anti-viral drug target can be made so thatdrug-sensitive viral genomes dominantly inhibit the outgrowth ofdrug-resistant viral genomes in a cell. The ability of relatively unfitviruses to inhibit growth of viruses with increased fitness derivesfrom, for example, the intracellular amplification of positive-strandRNA-viral genomes, their translation into large polyproteins, and thehigher-order oligomerization of several of their protein products.

Accordingly, the invention is based in part on the concept that certainviral products, when defective, can dominantly interfere with growth ofnon-defective viruses, making these viral products good drug targets.Interaction of an anti-viral drug with a viral product essentiallyrenders that viral product “defective” in function. Thus, if a viralproduct that, when defective due to mutation or drug interaction, istargeted by an anti-viral drug, then upon such viral products willdominantly interfere with other viruses in the cells in the presence ofthe drug. Stated differently, if defects similar to those introduced bya dominant mutations can be mimicked by antiviral drugs, the defectivedrug-sensitive viral genomes and products will dominantly interfere withthe intracellular growth of any resistant mutant genomes that mightarise.

Accordingly, drug sensitive viruses (i.e., viruses that produce a viraldrug target that, upon drug interaction, dominantly interferes withother viruses) will be dominant over drug-resistant variants whichcontain a mutation in the targeted viral product. Therefore, as mutant,drug-resistant viruses arise in the cell, the presence of other virusesin the same cell that are still drug-sensitive will inhibit the growthof the drug-resistant virus. As such, this strategy will at least delaythe emergence of drug-resistant viruses. This concept was borne out inexperiments conducted with poliovirus, a positive-strand RNA virus.

Suitable drug targets were identified by introducing mutations into thepoliovirus genome, generating variant, non-viable poliovirus, a numberof which encoded variant viral protein. Mammalian cells were cultured invitro, where the cells included both parent poliovirus and variantpoliovirus. A number of variant poliovirus that interfered with growthof the parent poliovirus in the cell were identified, and the mutationsin the genomes of the variant poliovirus were identified. Mutationsgiving rise to the growth-interfering phenotype included mutationswithin the capsid coding region; mutations in the polymerase codingregion; mutations in the protein primer 3B coding region and in thecis-acting replication element; and mutations in the 2A proteinasecoding region. Because the variant proteins and/or RNAs interfered withgrowth of parent poliovirus, they were referred to as “dominanttargets.” A dominant target would be a RNA or protein target that, whendefective, dominantly interferes with the growth of non-defective virus.

It was also observed that the presence of a drug-sensitive virusinhibits growth of a drug-resistant virus in a cell in the presence ofthe drug, where the drug target is a dominant viral protein. FIG. 8provides a schematic depiction of an exemplary assay for the effect ofgrowth of a parent, drug-sensitive virus on growth of a variant,drug-resistant virus.

The present invention provides methods for identifying a candidateanti-viral agent, where the emergence of drug-resistant virus in anindividual following administration of such an agent is reduced ordelayed. A delay in emergence of drug-resistant virus allows anindividual to mount an effective immune response to the virus. Thus, theindividual can clear the infection before emergence of drug-resistantvirus.

Methods of Identifying Candidate Anti-Viral Agents

The present invention provides methods of identifying a candidateanti-viral agent. The methods generally involve:

a) culturing a mammalian cell in vitro, where the mammalian cellcomprises: i) a parent RNA virus, where growth of the parent RNA virusis inhibited by a test agent; and ii) a variant of the parent RNA virus,where growth of the variant RNA virus is resistant to the test agent;and

b) determining the effect, if any, of parent virus growth on growth ofthe variant virus during at least one replicative cycle.

If parent virus growth interferes with the variant virus growth duringat least one replicative cycle, the test agent is considered a candidateanti-viral agent. In some embodiments, the parent virus growthinterferes with growth of the variant virus during one replicative cyclein vitro or in vivo. In other embodiments, the parent virus growthinterferes with growth of the variant virus during two, three, four,five, six, seven, eight, nine, ten, or more, replicative cycles in vitroor in vivo. Mammalian cells cultured in vitro and comprising both parentRNA virus and variant RNA virus, e.g., step (a), can be cultured in thepresence or absence of the test agent. In some embodiments, step (a) iscarried out in the absence of test agent.

A replicative cycle varies from virus to virus; replicative cycles forvarious RNA viruses are well known to those skilled in the art. Forexample, a single replicative cycle for poliovirus is about 6 hours in amammalian cell in in vitro cell culture; and a single replicative cyclefor HCV is about 48 hours in a mammalian cell in in vitro cell culture.

Whether a parent virus interferes with growth of a variant virus duringat least one replicative cycle is readily determined using knownmethods. For example, growth of the parent virus and growth of thevariant virus is determined in in vitro culture. The parent virus andthe variant virus are introduced into a mammalian cell in in vitroculture; and the effect, if any, of growth of the parent virus on growthof the variant virus is determined.

In some embodiments, the effect of growth of the parent RNA virus ongrowth of the variant virus is determined by introducing both parent RNAvirus and variant RNA virus into mammalian cells in in vitro culture,where the parent RNA virus is introduced into mammalian cells at amultiplicity of infection (MOI; or the average number of virus genomesper cell) of from about 1 to about 100, e.g., the parent RNA virus isintroduced into mammalian cells at an MOI of from about 1 to about 5,from about 5 to about 10, from about 10 to about 20, from about 20 toabout 30, from about 30 to about 40, from about 40 to about 50, fromabout 50 to about 70, or from about 70 to about 100; and the variant RNAvirus is introduced into the mammalian cells at an MOI of from about0.01 to about 1, e.g., the variant RNA virus is introduced into themammalian cells at an MOI of from about 0.01 to about 0.05, from about0.05 to about 0.07, from about 0.07 to about 0.1, from about 0.1 toabout 0.2, from about 0.2 to about 0.5, or from about 0.5 to about 1.0.In some embodiments, the ratio of MOI of the parent virus to the variantvirus is from about 5 to about 10⁴, e.g., from about 5 to about 10, fromabout 10 to about 10², from about 10² to about 5×10², from about 5×10²to about 10³, from about 10³ to about 5×10³, or from about 5×10³ toabout 10⁴.

Growth of the drug-sensitive, parent RNA virus inhibits growth of thedrug-resistant, variant RNA virus, e.g., the yield of drug-resistant,variant RNA virus is reduced. For example, growth of the drug-sensitive,parent RNA virus reduces the yield of drug-resistant, variant RNA virusper cell in in vitro mammalian culture by at least about 10%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or at least about 90%, or more (e.g., atleast about 95%, at least about 98%), during at least one replicativecycle (e.g., during one, two, three, four, five, or more replicativecycles). In some embodiments, yield of the drug-resistant, variant RNAvirus is undetectable during one or more replicative cycles.

In some embodiments, step (a) is carried out in the absence of the testagent; and determining the effect of growth of the parent RNA virus onthe variant virus is carried out using a plaque assay in the presenceand in the absence of the test agent. In the absence of the test agent,the plaque assay provides the total number of viruses (parent+variant);and in the presence of the test agent, the plaque assay provides thenumber of variant virus. A control can be carried out, in whichmammalian cells are culture in vitro, which mammalian cell comprise onlythe variant RNA virus; and the number of variant virus determined usinga plaque assay in the presence and absence of the test agent. An exampleof such an assay is depicted schematically in FIG. 8.

Virus growth in vitro is readily measured using known assays. Forexample, in some embodiments, virus produced by a cell is harvested andvirus stocks are determined by plaque assay. As another example, theparent virus and the variant virus could be constructed such that theparent virus includes a first reporter gene operably linked to apromoter; and the variant virus includes a second reporter gene operablylinked to a promoter, where the first and second reporter genes encodeproducts that are distinguishable from one another; and the effect ofparent virus growth on variant virus growth is determined by detectingthe first and second gene products. As another example, viral RNA ismeasured within the cell or in cell extracts or supernatants, virionproduction is measured by viral capsid or envelope protein in thesupernatant.

Suitable reporter gene products include, but are not limited to,luciferase (e.g., firefly luciferase and derivatives thereof; Renillaluciferase and derivatives thereof); β-galactosidase; chloramphenicolacetyl transferase; glutathione S transferase; a green fluorescentprotein (GFP), including, but not limited to, a GFP derived fromAequoria victoria or a derivative thereof, a number of which arecommercially available; a GFP from a species such as Renilla reniformis,Renilla mullen, or Ptiosarcus guernyl, as described in, e.g., WO99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519; any of avariety of fluorescent and colored proteins from Anthozoan species, asdescribed in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973,U.S. Patent Publication No. 2002/01 97676, or U.S. Patent PublicationNo. 2005/0032085; a red fluorescent protein; a yellow fluorescentprotein; a LUMIO™ tag (e.g., a peptide which is specifically bound by afluorescein derivative having two As(III) substituents, e.g.,4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein; see, e.g., Griffin etal. (1998) Science 281 :269; Griffin et al. (2000) Methods Enzymol.327:565; and Adams et al. (2002) J. Am. Chem. Soc. 124:6063); and thelike.

Growth of the drug-sensitive, parent RNA virus inhibits growth of thedrug-resistant variant RNA virus, e.g., the yield of drug-resistant,variant RNA virus is reduced. However, in some embodiments, poduction ofvariant viral RNA in the cytoplasm is unaffected by growth of the parentRNA virus. Whether production of variant virus RNA is reduced by growthof the parent RNA virus is readily determined using known assays. Forexample, RNA can be harvested from cells, and the amounts of parent andvariant virus determined using a reverse-transcription/polymerase chainreaction (RT-PCR) method.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7Cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721),COS Cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells(ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, and the like.

Parent RNA virus and variant RNA virus are introduced into the mammaliancell in vitro using any of a variety of methods, a number of which arewell known in the art. For example, where the virus is an encapsidatedvirion, the virus can be introduced into a mammalian cell by infection.Where the virus is not encapsidated (e.g., the virus is in the form ofnon-encapsidated RNA or other non-encapsidated nucleic acid), the viruscan be introduced into a mammalian cell by electroporation, transfectionusing DEAE-dextran, lipofection, and the like. DNA plasmids that encodethe viral RNAs can also be used.

Virus growth in vivo is readily measured using known assays. Forexample, a mammal (e.g., a rodent) is infected with both parent andvariant viruses, and virus growth is measured at various timespost-infection. Virus may be harvested from specific tissues or organs(e.g. skin, brain, muscle, spleen, etc.) and the tissue disrupted (e.g.by sonication, freeze-thaw followed by mortar/pestle grinding,homogenization using blender or dounce, etc) and the emerging virusquantified by plaque assay or reporter gene assay as described above.Virus growth can be measured by plaque assay or by detection of viralproducts in the cell supernatant or by detection of intracellular viralgenomes.

Parent RNA Virus

A parent RNA virus that is suitable for use in a subject method exhibitssensitivity to one or more test agents, e.g., a test agent inhibitsgrowth of the parent RNA virus. Parent RNA viruses that are suitable foruse in a subject method include wild-type RNA virus; wild-type RNAgenome; any known serotype of an RNA virus; a DNA copy of a wild-typeRNA genome; replication-competent variants of a wild-type RNA virus thatretain sensitivity of a wild-type virus to a selected test agent and iscapable of replication in a mammalian host cell; recombinant constructscomprising an RNA virus genome, or a DNA copy of an RNA virus genome;and sub-genomic replicons that retain sensitivity of a wild-type virusto a selected test agent.

Suitable parent RNA viruses include positive-strand RNA viruses andnegative-strand RNA viruses. Suitable positive-strand RNA virusesinclude, but are not limited to, members of Picornaviridae,Flaviviridae, Togaviridae, Caliciviridae, Coronaviridae, andRetroviridae families. Positive-strand RNA viruses generally (exceptretroviruses) share the following features: replicate in the cytoplasm;genomic RNA serves as a message and is translated; genomic RNA isinfectious; virions do not contain any enzymes; and viral proteins aretranslated as polyproteins.

Picornaviridae family members include, but are not limited to, membersof genus Enterovirus (including poliovirus, enterovirus, coxsackievirus,echovirus); members of the genus Rhinovirus; members of the genusHepatovirus (hepatitis A virus); encephalomyocarditis virus (EMCV); andfoot-and-mouth disease virus (FMDV).

Flaviviridae family members include, but are not limited to, members ofthe genus flavivirus, e.g., Dengue virus, Yellow Fever Virus, St. Louisencephalitis virus, Japanese encephalitis virus, and West Nile virus;members of the genus hepacivirus, e.g., hepatitis C virus; and membersof the genus pestivirus, e.g., bovine viral diarrhoea virus (BVDV).

Togaviridae family members include members of the genus alphavirus,e.g., Eastern encephalitis, western encephalitis, Sindbis, and Semlikiforest viruses; and members of the genus rubivirus, e.g., rubella virus.

Retroviridae family members include members of the genus lentivirusesincluding, but not limited to, human immunodeficiency virus, simianimmunodeficiency virus (SIV), and feline immunodeficiency virus.

Suitable negative-strand RNA viruses include, but are not limited to,Filoviridae family members (including Marburg virus, Ebola virus);Orthomyxoviridae family members (including influenza virus);Paramyxoviridae family members (including measles virus), andRhabdoviridae family members (including rabies virus).

In some embodiments, a parent virus is a recombinant constructcomprising an RNA virus genome, or a DNA copy of an RNA virus genome.The entire RNA genome (or a DNA copy thereof) may be present; however,the entire RNA genome (or a DNA copy thereof) need not be present in therecombinant construct. Suitable constructs include plasmid constructsthat include a cDNA copy of an RNA virus genome, or a sub-genomicportion of an RNA virus genome. Sub-genomic replicons of HCV are knownin the art and include, e.g., those described in U.S. Pat. No.6,956,117.

Poliovirus is exemplified in the Examples, below. Nucleotide and aminoacid sequences of poliovirus are known in the art. For example, thenucleotide sequence of poliovirus type 1 (Mahoney strain) is set forthin GenBank Accession No. NC_(—)002058; and is presented as SEQ ID NO:1;and amino acid sequences of the encoded proteins are set forth inGenBank Accession No. NP_(—)041277; and presented as SEQ ID NO:2. Aminoacid sequences of individual proteins encoded by poliovirus type 1(Mahoney) are provided in GenBank Accession Nos. NP_(—)740469 (VP2; SEQID NO:3; and FIG. 9); NP_(—)740470 (VP3; SEQ ID NO:4; and FIG. 10);NP_(—)740471 (VP1; SEQ ID NO:5; and FIG. 11); NP_(—)740477 (2A; SEQ IDNO:6; and FIG. 12); NP_(—)740472 (2B; SEQ ID NO:7; and FIG. 13);NP_(—)740473 (2C; SEQ ID NO:8; and FIG. 14); NP_(—)740474 (3A; SEQ IDNO:9; and FIG. 15); NP_(—)740475 (3B; SEQ ID NO:10; and FIG. 16);NP_(—)740476 (3C; SEQ ID NO:11; and FIG. 17); NP_(—)740478 (3D; SEQ IDNO:12; and FIG. 18); and NP_(—)740468 (VP4; SEQ ID NO:13 and FIG. 19).Sequences of poliovirus type 3 (Fox strain) are set forth in GenBankAccession No. AY359875. Sequences of poliovirus type 3 (Sabin) are setforth in GenBank Accession Nos. X00596 and P03302.

Variant RNA Virus

A variant RNA virus is a variant of any of the above-described parentRNA virus that, unlike the parent RNA virus, is resistant to aparticular agent, e.g., the variant virus grows in a mammalian cell inthe presence of a selected test agent. Thus, e.g., where growth of aparent RNA virus is inhibited by a selected test agent, growth of avariant of the parent RNA virus is resistant to the test agent.

A variant RNA virus genome comprises one or more changes in nucleotidesequence relative to the nucleotide sequence of the parent RNA virusgenome. The one or more changes in nucleotide sequence result in achange in the RNA and/or an encoded protein that inhibits growth of theparent virus RNA in a mammalian cell containing both parent virus RNAand variant virus RNA when the cell is cultured in the absence of a testagent. Thus, the one or more changes in nucleotide sequence result in achange that provides for a dominant-negative phenotype when parent virusand a variant virus are present together in the same mammalian cell inthe absence of any test agent. In some embodiments, the one or morechanges in nucleotide sequence result in a change in viral RNA orviral-encoded protein such that the variant virus, is non-viable whengrown in the absence of parent virus (or any other wild-type virus orany other virus that compensates for the mutation) in a mammalian cell.

As noted above, the one or more changes in nucleotide sequence result ina change that provides for a dominant-negative phenotype when parentvirus and a variant virus are present together in the same mammaliancell in the absence of any test agent. Thus, e.g., when variant RNAvirus and parent virus are introduced into mammalian cells in in vitrocell culture in the absence of test agent, and at an excess of variantvirus over parent virus, the variant virus reduces parent viral growthby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or at leastabout 90%, or more (e.g., at least about 95%, at least about 98%),during at least one replicative cycle.

In some embodiments, the variant RNA virus comprises one or more changesin nucleotide sequence, compared to the nucleotide sequence of theparent RNA virus, where the one or more nucleotide sequence changesalter the amino acid sequence of an encoded viral oligomeric protein,such that the virus produces a variant viral oligomeric protein in thecytoplasm of a mammalian cell. Variant viral oligomeric proteins includevariants having one or more amino acid sequence changes relative to theparent oligomeric protein, where amino acid sequence changes includeinsertions, deletions, truncations, substitutions, etc. In someembodiments, a variant viral oligomeric protein has a single amino acidsubstitution, compared with the parent viral oligomeric protein. Thevariant oligomeric protein inhibits growth of a parent virus present inthe same cell. Oligomeric viral proteins that, when mutated, exhibit adominant negative phenotype, include, but are not limited to, viralcapsid proteins; membrane-associated proteins; and polymerases (e.g.,RNA-dependent RNA polymerase).

In some embodiments, the variant RNA virus comprises one or more changesin nucleotide sequence, compared to the nucleotide sequence of theparent RNA virus, where the one or more nucleotide sequence changesalter the amino acid sequence of an encoded viral trans-acting protein.Variant viral trans-acting proteins include variants having one or moreamino acid sequence changes relative to the parent trans-acting protein,where amino acid sequence changes include insertions, deletions,truncations, substitutions, etc. In some embodiments, a variant viraltrans-acting protein has a single amino acid substitution, compared withthe parent viral trans-acting protein. The variant trans-acting proteininhibits growth of a parent virus present in the same cell. Trans-actingproteins include, but are not limited to, protein primer; polymerases,proteinases that cleave polyproteins; RNA helicases; membrane associatedproteins, and the like.

Identification of Dominant Negative Protein Targets

Suitable viral variants can be identified using a method as follows. Insome embodiments, a target for anti-viral drug intervention isidentified by a method comprising: a) introducing a mutation into acoding region of the genome of a parent RNA virus, generating a variantRNA virus comprising a variant coding region; b) co-transfecting amammalian cell in vitro with the parent RNA virus and the variant RNAvirus; and c) determining the effect, if any, of the variant RNA viruson growth of the wild-type RNA virus. Inhibition of growth of the parentRNA virus by the variant RNA virus indicates that the product of thevariant coding region is a suitable target for anti-viral drugintervention.

Methods for introducing mutations into a coding region of a viral genomeare well known in the art, and any known method can be used. Mutationsthat are introduced into the viral genome result in one or more changesin amino acid sequence of an encoded viral protein, relative to theamino acid sequence of the viral protein encoded by the parent RNAvirus. Mutations include random mutations and directed (non-random)mutations.

Methods of mutating a nucleic acid are well known in the art and includewell-established chemical mutation methods, radiation-inducedmutagenesis, and methods of mutating a nucleic acid during synthesis.Chemical methods of mutating DNA include exposure of DNA to a chemicalmutagen, e.g., ethyl methanesulfonate (EMS), methyl methanesulfonate(MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N′-nitrosoguanidine,4-nitroquinoline N-oxide, diethylsulfate, benzopyrene, cyclophosphamide,bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard,vincristine, diepoxyalkanes (e.g., diepoxybutane), ICR-170,formaldehyde, procarbazine hydrochloride, ethylene oxide,dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil,hexamethylphosphoramide, bisulfan, and the like. Radiationmutation-inducing agents include ultraviolet radiation, γ-irradiation,X-rays, and fast neutron bombardment. Mutations can also be introducedinto a nucleic acid using, e.g., trimethylpsoralen with ultravioletlight. Random or targeted insertion of a mobile DNA element, e.g., atransposable element, is another suitable method for generatingmutations. Mutations can be introduced into a nucleic acid duringamplification in a cell-free in vitro system, e.g., using a polymerasechain reaction (PCR) technique such as error-prone PCR. Non-randommutations can be introduced using a PCR method, where a primer is usedthat has one or more nucleotide differences from the template. Mutationscan be introduced into a nucleic acid in vitro using DNA shufflingtechniques (e.g., exon shuffling, domain swapping, and the like).Mutations can also be introduced into a nucleic acid as a result of adeficiency in a DNA repair enzyme in a cell, e.g., the presence in acell of a mutant gene encoding a mutant DNA repair enzyme is expected togenerate a high frequency of mutations (i.e., about 1 mutation/100genes-1 mutation/10,000 genes) in the genome of the cell. Examples ofgenes encoding DNA repair enzymes include but are not limited to Mut H,Mut S, Mut L, and Mut U, and the homologs thereof in other species(e.g., MSH 1-6, PMS 1-2, MLH 1, GTBP, ERCC-1, and the like). Methods ofmutating nucleic acids are well known in the art, and any known methodis suitable for use. See, e.g., Stemple (2004) Nature 5:1-6; Chiang etal. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl.Acad. Sci. USA 91:10747-51; and U.S. Pat. Nos. 6,033,861, and 6,773,900.

The variant viral-encoded protein will have one or more changes in aminoacid sequence, compared to the amino acid sequence of the same proteinencoded by the parent RNA virus. Variants will have one or more aminoacid substitutions, insertions, or deletions compared to the amino acidsequence of the same protein encoded by the parent RNA virus. In someembodiments, the variant viral-encoded protein will have a single aminoacid substitution, compared to the amino acid sequence of the sameprotein encoded by the parent RNA virus. Variant RNA sequences will haveone or more nucleotide changes relative to parent RNA virus.

Test Agents

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Test agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible. The term “test agent”excludes WIN-51711.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. In some embodiments, test agents include neutralizingantibodies. In some embodiments, neutralizing antibodies arespecifically excluded.

Selecting for Drug-Resistant Viral Variants

As noted above, a subject method for identifying a candidate anti-viralagent involves culturing a mammalian cell in vitro, where the mammaliancell comprises a drug-sensitive parent RNA virus (e.g., a parent RNAvirus that exhibits growth inhibition in the presence of a test agent)and a drug-resistant variant RNA virus (e.g., a variant virus, wheregrowth of the variant virus is resistant to the test agent). Selectionof a variant RNA virus that is resistant to a test agent can be carriedout using standard methods. For example, mammalian cells comprising aparent RNA virus are cultured in vitro in the presence of a test agentthat inhibits growth of the parent RNA virus. Variants are selected thatare not growth-inhibited, and therefore are resistant to the test agent.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Identification of Dominant Targets

Methods

Design and Characterization of Mutant Genomes Used for DominantInhibitor Screen.

To amass a collection of nonviable mutant poliovirus genomes, previouslycharacterized lethal mutations, or mutations that were designed todestabilize the structure of the encoded viral protein product, wereintroduced using two strategies. The first strategy was to disrupt thehydrophobic core of the encoded protein by reducing the size of an aminoacid side chain predicted to be inaccessible to solvent or by changingit to Pro. A second strategy was to disrupt predicted α-helices byaltering Leu and Ser residues predicted to reside in α-helices to Pro. Acomputer algorithm, PredictProtein³³, was used to predict the likelysolvent accessibility and α-helicity of each amino acid position withinthe poliovirus type 1 (Mahoney) polyprotein in its native, folded state.Single amino acids presumed to be within the hydrophobic protein core byvirtue of displaying a PredictProtein score greater than five werealtered by introducing a single U-to-C transition mutation into theviral genomes, using PCR-mediated site-directed mutagenesis³⁴. ThePredictProtein method was used instead of known three-dimensionalstructures to simulate the knowledge base of other, lesswell-characterized, positive-strand RNA viruses.

The viability of each mutant genome was tested by transfecting 60 mmplates of subconfluent S3 HeLa monolayers with 1 μg of in vitrotranscribed mutant RNA using DEAE-dextran (see below). Viral stocks wereharvested after a single replicative cycle (10 hrs at 32.5° C.) bypelleting the cells at 200×g, washing and lysing by freeze-thaw in 1 mlof PBS+ (phosphate buffered saline with 0.1% CaCl₂ and MgCl₂), andlysing the cells by freeze-thaw treatment. Stocks were then titered aspreviously described³⁵. Mutant genomes that produced no detectableplaques in this assay were defined as nonviable; 1 μg of wild-type RNAtypically yielded between 4×10⁵-1×10⁶ PFU under these conditions. Theresults of the engineered and previously characterized mutations aredescribed in Tables 1 and 4. Reversion of mutant genomes to producewild-type virus was not detected using DEAE-dextran transfections.

RNA Transcription and Co-Transfections

Plasmids containing the poliovirus 1 genome (pGEM-PV1) under the controlof a T7 promoter were linearized by Eco R1 restriction digestion (NewEngland Biolabs, Beverly, Mass.), purified by agarose gelelectrophoresis, and used as a template for transcription using theRibomax (Promega, Madison, Wis.) T7 transcription kit according to themanufacturer's standard protocol. A control RNA, R2-PvuII (see below),was made from pGEM-PV1 cDNA that lacks capsid encoding nucleotides 1175to 2956 and linearized with PvuII, which cleaves within the codingregion of 3D polymerase. All transcription reactions were extractedtwice with acid phenol-chloroform-(5:1, pH 4.5, Ambion) to removeprotein and template DNA, and precipitated with a half volume of 7.5 Mammonium acetate and 2.5 volumes ethanol. Precipitated pellets of RNAwere then resuspended in water and applied to a P30 size exclusioncolumn (Biorad, Hercules, Calif.) to remove unincorporated nucleotidesand the eluate collected according to the manufacturer's protocol. Theintegrity of transcribed RNA was verified by denaturing formaldehydeagarose gel electrophoresis and the concentration determined bymeasuring absorbance at 260 nm. The preparations were reprecipitated,aliquoted and stored at −80° C. Transcription reactions were alsoperformed in the presence of α³²P-UTP to verify that RNA amountsmeasured by O.D. at 260 nm represented RNA transcripts and notunincorporated nucleotides.

The effect of each mutant genome on the growth of wild-type virus wastested by co-transfecting 60 mm plates of subconfluent S3 HeLamonolayers with 1 μg of in vitro transcribed mutant RNA and 100 ng ofwild-type poliovirus RNA using DEAE-dextran (average molecularweight=500,000; Sigma, St. Louis, Mo.) as described previously⁸. After10 hours incubation of each transfection at 32.5° C., cells wereharvested, virus stocks produced, and plaque assays performed asdescribed³⁵.

In Vitro Translation of Poliovirus RNAs and α-VP1 Immunoprecipitation ofTranslation Reactions

Poliovirus RNAs were transcribed as for transfections. HeLa S10 extractswere prepared as previously described³⁶, and a 25 μl reaction wasprogrammed with 5 μg of RNA, 50% HeLa extract, 1 μl ³⁵S-Met Expresslabel (NEN), 0.5 μl RNasin (Promega), and 0.5 μl 1 mM of each amino acidexcept methionine. Each reaction was incubated for 2 hours at 30° C.,and then stopped by addition of 2×-lysis buffer (2% Triton X-100prepared in TBS: 10 mM Tris-Cl pH 8.0, 140 mM NaCl, and 0.025% NaN₃).Each reaction was then centrifuged at 10,000×g for 30 minutes at 4° C.To clear the supernatant, 1/20^(th) volume of a 50% slurry of protein-Gsepharose beads (Gibco-BRL, Grand Island, N.Y.) was added to thesupernatant, incubated at room temperature for 2 hrs, and pelleted by 1min. centrifugation at 200×g. The supernatant was transferred to a newtube precoated with lysis buffer that contained 200 μl of dilutionbuffer (0.1% Triton X-100 prepared in TBS). Monoclonal mouse α-VP1antibody (3 μg; Chemicon, Temecula, Calif.) was added to each reactionand incubated 1.5 hours at 4° C. The beads were pelleted at 200×g andwashed twice with dilution buffer, once with TBS, and once with 50 mMTris-Cl (pH 6.8). Proteins in the samples were then separated on a 10%polyacrylamide gel by SDS-PAGE analysis.

Super-Infections

Temperature-sensitive viruses were either previously described²⁴ orgenerated during the screening of nonviable poliovirus type 1 (Mahoney)mutants (Table 3). HeLa cell monolayers were infected at an MOI=100PFU/cell with each temperature-sensitive virus at a semi-permissivetemperature of 37° C. in PBS+. After virus adsorption (30 minutes),serum supplemented DMEM was added with 2 mM guanidine hydrochloride toblock viral RNA synthesis. At two hours post-infection, the media wasremoved and cells were super-infected with wild-type virus at an MOI of0.5 PFU/cell. Incubation at 37° C. was continued for 4 hours in theabsence of guanidine, whereupon cells were harvested and virus stockswere made as described above. Virus titers were determined at 39° C.

Co-Infections of WIN-Sensitive and WIN-Resistant Viruses

WIN-resistant Sabin 3 virus was engineered by introducing a mutation,VP1-I192F, into a cDNA encoding the attenuated Sabin type 3 poliovirus.Infections and co-infections of HeLa cell monolayers at the indicatedMOI's for 30 minutes at 37° C. Serum-supplemented DMEM (10%) with orwithout 2 μg/ml final concentration WIN-51711 was added to the cells,and incubation continued at 37° C. for 6 hours. Cells were thenharvested and virus stocks titered by plaque assay in the absence andpresence of 2 μg/ml WIN-51711 in the agar overlay.

RT-PCR of WIN-R and Wild-Type RNA

Co-infections and single infections of wild-type and WIN-resistantviruses were performed as described above in the absence of WIN-51711.At 5 hours post-infection, cells were harvested and processed by theaddition of 1 ml Trizol (Invitrogen, Carlsbad, Calif.) to each plate andincubation for 5 min. at room temperature. The solution was extractedwith 0.2 ml chloroform and the supernatants were collected. At thistime, “mix” samples were created by combining equal volumes ofsupernatants derived from single infections (see FIGS. 6 c,d) Nucleicacids were collected by the addition of isopropanol (70%), pelleting bycentrifugation, washing with 70% ethanol, repelleting, and resuspensionin 50 μl 10 mM HEPES-KOH (pH 7.5).

Each reverse transcriptase reaction contained 5 μl RNA sample in a finalvolume of 10 μl using AMV-RT High Concentration (Promega, Madison, Wis.)as recommended by the manufacturer. PCR reactions (50 μl total volume)were composed of 5 μl of a reverse transcriptase reaction and performedas previously described³⁷. Reactions were cycled 35 times (94° C., 1minute; 54° C., 1 minute; 72° C. 1 minute). Primers used amplified aregion from nucleotide 2967 to 3241 of the type 3 genome. Two TfiIrestriction sites (at nucleotides 3048 and 3149) exist within thewild-type PCR product, but the 3048 site is disrupted by the VP1 I192FWIN-R mutation. PCR reactions were brought to 200 μl volumes anddigested with 25 units TfiI for two hours at 65° C. Reactions were thenethanol precipitated and analyzed by PAGE on a 5% polyacrylamide, 8Murea gel.

Results

Design and Characterization of Nonviable Poliovirus Mutations

To search for dominant alleles in a comprehensive, genome-wide manner, abattery of lethally mutated genomes spanning the poliovirus codingregion was constructed (Table 1). The construction of each mutant genomewas guided either by a previously described mutation or by a strategy todisrupt the structure of the encoded protein. By targeting predictedhydrophobic cores or α-helices (see Methods), 24 individual U-to-Cmutations were introduced into an infectious poliovirus cDNA and theviability of each mutant viral genome was tested. The results of thesetransfections and the rationale for each mutation are shown in Table 1.

TABLE 1 Mutant poliovirus genomes constructed for use in the dominancescreen. Codon Viability Mutant Change Rationale (PFU/ml) Previouslycharacterized mutant poliovirus genomes VP2-S1P UCG→CCG Maturationcleavage (Ansardi <5 and Morrow 1995) VP2-S243P UCC→CCC Reynolds et al.1991 <5 2A-C109R UGU→CGU Catalytic proteinase cysteine <5 (Yu and Lloyd1991) 3B-Y3H UAC→CAC Uridylylation site (Rothberg <5 et al. 1978; Ambrosand Baltimore 1978) 3C-C147R UGU→CGU Catalytic proteinase cysteine <5(Hammerle et al. 1991) 3D-F30S UUC→UCC Fingers-thumb interaction <5(Hobson et al. 2001; Hansen et al. 1997) 3D-S291P UCA→CCA Burns et al.1989 <5 CRE-C4465U/ None Goodfellow et al. 2000 <5 U4483C* CRE-G4462A/None Goodfellow et al. 2000 <5 U4483C* Designed mutant poliovirusgenomes VP2-F260S UUC→UCC Hydrophobic <5 VP3-F118S UUU→UCU Hydrophobic<5 VP3-L211S CUU→CCU Hydrophobic <5 VP1-L118P UUA→UCA Hydrophobic &Helix <5 2A-S74P UCC→CCC Helix t.s. 2A-L98P CUC→CCC Hydrophobic <52A-F133S UUU→UCU Hydrophobic <5 2B-F13S UUU→UCU Hydrophobic & Helix <52B-F17S UUU→UCU Hydrophobic & Helix <5 2C-F28S UUC→UCC Hydrophobic &Helix t.s. 2C-L93P CUU→CCU Hydrophobic <5 2C-F242S UUU→UCU Hydrophobic<5 2C-F328S UUU→UCU Hydrophobic & Helix <5 3A-L8S UUG→UCG Hydrophobic <53A-L24S UUG→UCG Hydrophobic & Helix <5 3A-F83S UUU→UCU Hydrophobic <53C-L70P CUU→CCU Hydrophobic <5 3C-L102S UUG→UCG Hydrophobic <5 3D-F34LUUU→CUU Hydrophobic t.s. 3D-L107P CUA→CCA Hydrophobic <5 3D-F191SUUU→UCU Hydrophobic & Helix <5 3D-F246S UUC→UCC Hydrophobic & Helix <53D-F296S UUU→UCU Hydrophobic & Helix <5 3D-Y326H UAU→CAU Hydrophobic <5*Mutants in the CRE (cis-acting replication element) are double mutantsbecause viruses containing either single mutation were viable. CREmutations are non-coding mutations in the 2C coding region.

For 21 of the 24 designed mutations, no virus was detected upon a singlecycle of growth after RNA transfection, indicating an absence ofreversion to wild-type virus under these transfection conditions. Thethree remaining mutations gave rise to viable viruses withtemperature-sensitive phenotypes that were characterized further (Table3). For both previously published and designed mutations, only mutantgenomes that displayed a 100,000-fold or greater reduction in plaqueformation after RNA transfection were determined to be nonviable andused in the screen for dominant negative poliovirus alleles, whichidentified four classes of strongly dominant alleles.

Dominant-Negative Alleles in Capsid Coding Regions

To test the ability of nonviable mutant genomes to affect wild-typeviral growth, nonviable and wild-type viral RNAs were co-transfectedinto HeLa cells, the intracellular virus was harvested after a singlereplicative cycle, and the resulting wild-type virus stocks weretitered. To both mimic a scenario in which a drug-resistant genomeemerges from a drug-sensitive population, and to optimizeco-transfection conditions, a ten-fold excess of the nonviable genomewas chosen. Total yeast tRNA was substituted for mutant RNA in thepositive wild-type control. Under these conditions, a transfection ofapproximately 2×10⁶ cells with 100 ng wild-type RNA typically yielded avirus stock of 50,000-200,000 PFU (plaque-forming units)/ml.

The effect of a known inhibitor of poliovirus RNA replication, an RNAtranscript (R2-PvuII) made in vitro from a poliovirus cDNA templatecleaved with Pvu II⁷, was tested to ensure that the transfectionprotocol used led to co-transfection of the wild-type and potentiallyinhibitory genomes. When co-transfected with wild-type viral RNA, aten-fold excess of R2-PvuII RNA inhibited wild-type growth (FIG. 1 b),as reported previously⁸. Although the mechanism by which R2-PvuII RNAinhibits the growth of wild-type RNA is not known, the greater than20-fold inhibition of wild-type growth observed argues that at least 95%of the cells that contained wild-type viral RNA also contained theco-transfected inhibitor RNA.

Co-transfection of several of the lethally mutated RNAs, for examplefs-2956 and 3A-L24S, either had little effect or caused a slightincrease in wild-type yield (FIG. 1 b). Although 3A-L24S genomescontained a lethal point mutation in one coding region, it is likelythat other functional trans-acting proteins produced from these mutantgenomes provide helper functions for the wild-type genomes. Theframe-shift control, fs-2956, occurs in the center of the VP1 codingregion, and produces a truncated wild-type capsid region withtermination of translation at a stop codon at nucleotide 3129. Thisconstruct appears to have also provided a helper function by theproduction of capsid proteins.

In contrast, all four genomes that contained lethal mutations within thecapsid coding region, VP2-S1P, VP2-S243P, VP3 L211S, and VP1-L118P,reduced wild-type viral growth approximately 10- to 20-fold (FIGS. 1a,c), which was the same extent of inhibition observed for the R2-PvuIIco-transfection control. On average, capsid mutant genomes inhibitedwild-type growth to 7% of wild-type growth alone.

FIGS. 1 a-d. Dominant inhibitor screen for capsid-coding genome regions.(a) Schematic of mutant genomes tested as dominant inhibitors ofwild-type virus growth. (b) Validation of dominant inhibitor screen forR2-PvuII, a known RNA inhibitor of poliovirus growth, and two mutantalleles, a frameshift mutation at nucleotide 2956 (fs-2956) and 3A-L24S,that each provide apparent helper function. The average of each set ofreplicate experiments (with standard error) is shown below each set ofreplicates normalized to the average of the wild-type poliovirus RNAwith carrier tRNA control. (c) Mutant capsid alleles mapped to thecrystal structure of capsid proteins (VP4 in cyan, VP1 in yellow, VP2 inmagenta, VP3 in salmon)⁴⁸. (d) The effect of co-transfecting theindicated RNAs with wild-type RNA on yield of wild-type poliovirus isshown as in b.

Allele-Specific Inhibition by Mutations in the 3D Polymerase CodingRegion

Given the known ability of the poliovirus RNA-dependent RNA polymeraseto oligomerize¹⁰, the dominance of five different non-viable allelesthat contained mutations in the polymerase coding region was tested(FIGS. 2 a,b). One allele, S291P, diminished wild-type viral growth to1% of the control (FIG. 2 c), and thus exerted a larger dominant effectthan the R2-PvuII negative control. Two other alleles, F30S and F191S,diminished wild-type growth to 29% and 13%, respectively, and thus wereco-dominant. Other alleles showed variable intermediate or helpereffects and were deemed recessive. Mapped onto the fully resolved 3Dpolymerase structure¹¹, the majority of mutated residues cluster in thehydrophobic core of the fingers domain, while 3D-F30S is located at theinterface between the “finger” and “thumb” domains (FIG. 2 b). Whileverifying that the screen adequately identifies residues involved inhydrophobic interactions, the variability in observed dominance ofmutant 3D polymerase alleles may reflect varying degrees of proteinstability or the oligomerization potential of 3D polymerase or itsprecursors. Alternatively, the allele-specificity of these alleles mayalso reflect their mixed effects on either 3D polymerase or itsprecursor, 3CD protease.

FIGS. 2 a-c. Effect of mutations in 3D polymerase on the yield ofwild-type virus during co-transfection. (a) Schematic diagram ofpoliovirus genomes indicate locations of coding regions for mutant 3Dpolymerase alleles. (b) Mutant alleles mapped to the three-dimensionalstructure of 3D polymerase¹¹. (c) Parallel co-transfection experimentswith wild-type RNA and viral genomes containing several differentmutations in the coding region for 3D polymerase are shown as in FIGS. 1a-d.

Dominant Mutations of the Protein Primer 3B and Cis-Acting ReplicationElement (CRE)

A surprising result came from the trans-dominant effects of mutations inthe CRE, the nominally cis-acting RNA sequence that templates VPguridylylation in vitro¹⁴, and 3B (the VPg coding region), shown in FIG.3 a. Two non-coding double mutations in the CRE, G19A/U40C andC22U/U40C, as well as a mutation of the genome-linked structuralprotein, 3B-Y3H (FIG. 3 b), strongly inhibited growth of co-infectingwild-type virus. The degree of inhibition was similar to, or greaterthan, that exerted by R2-PvuII, the co-transfection control, or any ofthe capsid alleles.

A “classic” dominant negative allele is one in which the function of aprotein or sequence element is disrupted while an associative property,such as a protein-protein or protein RNA interaction, is retained¹⁷. Forthe dominant negative alleles of the CRE, the stem-loop structure ispredicted to be maintained (FIG. 3 a)¹⁶. The introduction of eightnon-coding mutations into the CRE, however, is predicted to completelydisrupt stem-loop structure and presumably any structure-specificassociations¹⁸. This predicted loss-of-function CRE allele (“l.o.f.CRE”) did not inhibit wild-type growth (FIG. 3 c). Therefore, dominantinhibition by mutant CRE-containing genomes is allele-specific,presumably requiring an intact RNA stem-loop structure to form aninhibitory complex. Presumably such a complex would involve 3CD, aprecursor known to bind RNA sequences in the 5′ UTR as well as theCRE.^(15,16).

FIGS. 3 a-c. Dominant inhibitor screen in the CRE and 3B-coding regions.(a) A schematic diagram of the predicted secondary structure of thewild-type CRE, which resides in the coding region of 2C, with indicatedG19A, C22U, and U40C mutations used in the dominance screen. CRE mutantsG19A/U40C and C22U/U40C correspond to genomic nucleotide positions4462/4483 and 4465/4483, respectively, and are previously publishednon-coding mutations⁴⁷. 3B encodes the protein primer, VPg, to whichuridyl residues are attached at Tyr-3. Asterisks (*) denote multiplenon-coding nucleotides mutated to form the “l.o.f.”, or putative“loss-of-function” CRE used in c¹⁸. (b) Co-transfection experiments wereperformed mixing wild-type and mutant genomes that contain the indicatedmutant alleles. (c) Specificity of mutant CRE alleles. Co-transfectionsof wild-type and mutant genomes that contain the specified CRE alleleswere performed as in FIGS. 1 a-d. Mutations that specify the “l.o.f.”mutant genome is illustrated in a.

Allele-Specific Dominance in 2A Proteinase Coding Region

Two different mutations tested in the 2A proteinase coding region (FIGS.4 a,b) showed pronounced dominance (2A-L98P and 2A-C109R) when comparedto fs-2956 (FIG. 4 c). The dominant phenotype correlated with proteasedeficiency: FIG. 4 d shows the accumulation of uncleaved VP1-2Aprecursor for the 2A-L98P and 2A-C109R mutations, whereas wild-type andgenomes containing mutations in the capsid coding region didnot^(19,20). Further experiments using only the VP1-2A region expressedin vitro recapitulated this protease-deficient phenotype.

The dominance of protease-deficient 2A alleles was surprising at first,because 2A proteinase is known to be a monomeric enzyme with severalviral and cellular substrates. However, its activity at the VP1-2Acleavage site is thought to be obligately intramolecular, becausecleavage is unaffected by α-2A antibodies, and occurs more rapidly andwith more specificity than intermolecular 2A proteinasecleavage^(21,22). A 2A protein that lacked enzymatic activity couldtherefore yield an uncleaved VP1-2A fusion molecule that functioned as adominant negative inhibitor of wild-type growth, like a mutant capsidprotein. To test this hypothesis explicitly, the VP1-2A cleavage sitewas mutagenized to allow accumulation of the uncleaved VP1-2A productfrom mutant genomes, and studied its effect on the growth ofco-infecting wild-type virus. Two introduced mutations reported toabrogate 2A-mediated cleavage of the VP1-2A cleavage site, VP1-Y302P andVP1-T301R²², were also dominant (FIG. 4 e). Therefore, uncleaved VP1-2Ais toxic to co-infecting wild-type virus, and its accumulation in cellsinfected with 2A proteinase-deficient mutant viruses is a likelymechanism for its genetic dominance.

FIGS. 4A-E. Dominant inhibitor screen for 2A proteinase and VP1-2Acleavage site mutant alleles. (a) Schematic diagram of poliovirusgenomes indicate relative locations of coding regions for 2A and VP1,and the 2A proteinase and VP1-2A cleavage site mutants used in thedominant inhibitor screen. (b) 2A proteinase amino acid residuestargeted to generate nonviable mutations used in the dominance inhibitorscreen and superinfection assay are mapped onto the crystal structure of2A proteinase from human rhinovirus 2, a closely related homolog ofpoliovirus 2A proteinase⁴⁹. (c) Co-transfections were performed as inFIG. 1 and the resulting wild-type virus yields of replicate experimentsare shown. Normalization of these values as a percentage of thewild-type RNA with tRNA carrier control is shown at bottom with standarderror. (d) HeLa cytoplasmic lysates were programmed with the indicatedpoliovirus RNAs and labeled using ³⁵S-methionine (Methods),immunoprecipitated with a monoclonal anti-VP1 antibody, and separated ona 10% SDS-PAGE gel. VP1 and VP1-2A mobilities are marked. The asterisk(*) denotes a higher molecular weight species abundant in 2A-L98P and2A-C109R reactions. (e) Testing for dominance of genomes with mutatedVP1-2A sites was performed as in FIGS. 1 a-d.

Translation and RNA Replication Requirements for Dominance

Genomes that contain mutant capsid alleles are usually known to becompetent for RNA replication; therefore, the observed dominance ofgenomes with capsid mutations may be augmented by the replication of themutant genomes, resulting in the production of high concentrations ofmutant capsid proteins. To determine whether or not mutant capsidalleles require RNA replication to exert their dominant effect, a secondmutation, ΔGUA₃, was introduced into one of the dominant mutant genomes.The deletion of nucleotides 7418-7422 in the 3′-non-coding region (FIG.5 a) is known to severely diminish negative-strand RNA synthesis⁹. Asshown in FIG. 5 b, the doubly mutant genome VP2-S243P/ΔGUA₃ did notinhibit wild-type virus growth. Therefore, the dominance of theVP2-S243P allele, and probably the other capsid alleles as well,requires replication of the mutant RNA genome, presumably leading toincreased accumulation of the VP2-S243P mutant capsid proteins.

As with capsid alleles, 3D polymerase functions can be rescued in transby polymerase molecules encoded by the co-transfected wild-type genomes,thus enabling genomes harboring mutant 3D polymerase alleles toreplicate^(8,12,13). Whether the dominance of the 3D-S291P allelerequired RNA replication of its genome by introducing the ΔGUA₃ deletionwas tested. As shown in FIG. 5 c, the 3D-S291P allele was no longerdominant when RNA replication of its genome was inhibited. Like theinhibitory effects of mutant capsid proteins, the high concentration ofdefective polymerases provided by a replicating genome is needed toinhibit the growth of co-infecting wild-type virus.

To test whether the observed dominance of the 3B mutation required RNAreplication of the nonviable genome to exert dominance, the ΔGUA₃mutation was introduced. Like dominant capsid and polymerase alleles,the 3B-Y3H allele required replication of its RNA genome to exhibitdominant negative effects on wild-type growth (FIG. 5 d).

However, when the same experiment was performed with dominant negativeCRE allele C22U/U40C, the triple mutant C22U/U40C/ΔGUA₃ was stillinhibitory, arguing that the mutant CRE structure was toxic at lowerconcentrations than the defective capsid, polymerase, or VPg proteins(FIG. 5 e). That such a dominant negative effect could occur without RNAreplication is not without precedent since our dominant negative controlfor co-transfection, R2-PvuII, lacks a 3′-non-coding region and alsopresumably lacks the ability to replicate. To determine whether the CRERNA alone was the inhibitory moiety, it was undertaken to determinewhether a non-translatable CRE-C22U/U40C genome was dominant. To thisend, the initial methionine of the poliovirus polyprotein was mutated toan amber stop codon (VP4-M1stop), and introduced into a genomecontaining the C22U/U40C CRE allele. As shown in FIG. 5 f, the dominantnegative phenotype of the C22U/U40C CRE allele was eliminated whennormal translation of the genome was blocked, arguing that it is not theCRE RNA alone, but some complex formed upon translation of viralproteins that inhibits the growth of other viruses in the same cell.

FIGS. 5A-F. RNA replication or translation requirements for dominance ofmutant poliovirus alleles. (a) Schematic of mutant alleles mapped to thepoliovirus genome assayed for dominance requirements. ΔGUA₃ is a genomelacking nucleotides G₇₄₁₈UAAA₇₄₂₂; RNAs that contain this 3′-non-codingregion deletion are deficient for negative strand synthesis³⁶. TheVP4-M1stop mutation changes the initial methionine of VP4 to a UAG stopcodon. (b) Test of replication requirement for dominance of VP2-S243Pgenome. Co-transfections were performed in triplicate and are graphed asthe average of replicate co-transfections with error bars to indicatestandard error. (c) RNA replication requirements for 3D S291P. A mutantgenome containing both the 3D-S291P and ΔGUA3 deletion wasco-transfected with wild-type RNA and graphed as in b. Theco-transfection of wild-type and a genome containing only the ΔGUA3deletion is shown as a control. (d) RNA replication requirements for amutant allele of the RNA replication protein primer, 3B-Y3H.Co-transfections were performed and graphed as in b, above. “ΔGUA₃alone” indicates that no wild-type RNA was co-transfected, while “ΔGUA₃”indicates a co-transfection of that RNA with wild-type virus. “3BY3H/ΔGUA₃” indicates the co-transfection of a non-replicating RNA thatcontains both mutations with wild-type RNA. No virus was detected at thehighest dilution of the ΔGUA₃ virus alone, so the limit of detection isgraphed. (e) RNA replication requirements for mutant CRE alleledominance. Co-transfections were performed as in b. “C22U/U40C/ΔGUA3”refers to the doubly mutant, non-replicating genome that contains themutant CRE allele and 3′-non-coding region deletion. (f) Translationrequirements for dominance of mutant CRE allele C22U/U40C.Co-transfections of the indicated genomes containing mutant alleles areshown as in b. “VP4-M1stop/C22U/U40C” is a non-translating genome thatcontains all indicated mutations.

Effects in Other Nonstructural Protein Coding Regions

The effect of mutations in the 2B, 2C, 3A, and 3C coding regions onco-transfected wild-type viral genomes is shown in Table 2.

TABLE 2 Dominant inhibitor screen results of mutant 2B, 2C, 3A, and 3Calleles. PFU/ml Percentage (×10⁴) of control tRNA 23  100 ± 29% control*R2-Pvull 0.49 2 ± 1 2B F13S 2.5 11 ± 2  2B F17S 2.7 11 ± 5  2C L93P 1876 ± 15 2C F242S 5.0 21 ± 13 2C F328S 10 44 ± 13 3A L8S 2.0 9 ± 2 3AF83S 10 44 ± 7  tRNA 24  100 ± 19% control* R2-Pvull 2.0 8 ± 2 3C-L70P35 144 ± 32  3C-L102S 54 226 ± 31  3C-C147R 57 239 ± 26  2A-F133S 54 224± 35  *Independent sets of co-transfections are shown.

Of the three membrane-associated proteins 2B, 2C, and 3A, mutations in2B were found to be more consistently dominant, although theirsuppression of wild-type growth was not as pronounced as that of theR2-PvuII control. Partially dominant mutations in the 2B coding regionhave been reported previously²³, although the mechanism of dominanceremains unknown. Unlike mutations in 2A proteinase (FIG. 4), mutationsin 3C proteinase were either recessive or gave rise to viruses thatprovided a helper function (Table 2).

Superinfections of Temperature-Sensitive and Wild-Type Polioviruses.

To test whether the locus- and allele-specific dominance ofco-transfected genomes would also be observed with viable viruses, theability of temperature-sensitive (ts) viruses (FIG. 6 a) to inhibit thegrowth of wild-type virus was monitored (Methods). First, cells wereinfected with ts mutant viruses in the presence of an RNA replicationinhibitor, guanidine hydrochloride, to allow mutant viral proteins toaccumulate to similar concentrations for each of the ts mutant viruses.At a later time point, guanidine was removed, wild-type virus was added,and a single cycle of viral growth continued at a semi-permissivetemperature. The resulting virus stocks were then titered at anon-permissive temperature to quantify the yield of wild-type virus. Asshown in FIG. 6 b, two mutant viruses with mutations in the 2Aproteinase (S74P) and 2C NTPase (F28S) coding regions did not hinderwild-type growth. However, a virus that contained a mutation in thecapsid coding region²⁴ (VP2-R76Q) reduced wild-type virus growth by morethan 260-fold compared to wild-type growth alone (FIG. 6 c).

FIGS. 6A-C Superinfections of wild-type and temperature-sensitivepolioviruses. HeLa cells were infected with the temperature-sensitivepoliovirus indicated while blocking RNA synthesis as described inMaterials and Methods. After mutant proteins were allowed to accumulate,wild-type poliovirus was added to HeLa cells as indicated, and the blockto RNA synthesis released. Viral titers after a single cycle ofinfection are shown above for two separate experiments at therestrictive temperature. (a) Schematic of temperature-sensitive allelesused in superinfections. (b) Yield of wild-type virus followingsuperinfection of cells that had accumulated protein fromtemperature-sensitive mutant viruses 2A-S74P or 2C-F28S (Table 3) astemperature sensitive viruses. (c) Yield of wild-type virus followingsuperinfection of cell that had accumulated proteins fromtemperature-sensitive mutant viruses VP2-R76Q²⁴, 3D-F34L (Table 3), or3D-T367I²⁵.

Two viruses with different mutations in the coding region for the viralRNA-dependent RNA polymerase, 3D-F34L and 3D-T367I (Table 3)²⁵, weretested for their ability to hinder wild-type growth. While 3D-T367Ivirus was not inhibitory, 3D-F34L exhibited a four-fold inhibition ofwild-type virus growth, thus showing that dominance of mutant genomesdiffered between coding regions and between alleles of the same codingregion.

TABLE 3 Phenotypes of temperature-sensitive polioviruses generated byhydrophobic mutations. Phenotype PFU/μg PFU 39° C./ RNA 32.5° C. 39° C.32.5° C. wild- 290 large plaque large plaque 1.4 type 2A- 190 very smallnone detected <0.07 S74P plaque 2C- 140 small plaque small plaque 0.05F28S 3D- 990 small plaque very small 0.4 F34L plaqueSummary of Dominant Negative Alleles

A genomic screen with poliovirus, a positive-strand RNA virus, wasconducted to identify viral proteins that, when made nonfunctional bymutation, would dominantly interfere with the growth of co-transfectedwild-type viral RNA genomes. These proteins should constitute ideal drugtargets if the defects of the dominant alleles can be phenocopied by theantiviral compounds. Twenty-seven different genomes, each of whichcontained a single lethal mutation, were tested. Four classes ofstrongly dominant mutations were observed (Table 4). First, capsidmutations were dominant, presumably because nonfunctional mutant capsidsco-assemble with wild-type capsids and render them nonfunctional. Asecond class of dominant genomes contained mutations in the polioviruspolymerase coding region; however, only two out of seven mutations inthe polymerase coding region were strongly dominant. Polioviruspolymerase is known to oligomerize¹⁰; therefore, the mechanism ofdominance is likely to be similar to that of the capsid alleles. A thirdclass of strongly dominant mutations in poliovirus was found in an RNAstructure, termed the CRE, that is required for generation of theprotein primer for polioviral RNA synthesis. Finally, mutants thatrendered the 2A proteinase of poliovirus inactive (L98P and C109R, FIG.4) were dominant and profoundly inhibitory. The cleavage between VP1 and2A coding regions within the viral polyprotein is made by 2A proteinaseand reported to be intramolecular²¹, and would therefore be refractiveto scission in a mutant proteinase even in the presence of mature,wild-type 2A proteinase encoded by coinfecting genomes. Uncleaved VP1-2Aprotein encoded by the mutant genomes may inhibit co-infecting genomesin the same way that mutant capsid protein does, by co-assembling withwild-type capsids and poisoning their function. To test this hypothesis,the effects of directly mutating the VP1-2A cleavage site weredetermined. These mutants were also dominant.

TABLE 4 Summary of dominant negative alleles of poliovirus. PotentialMechanism Recessive or cis-Dominant Alleles^(a) 2A-F133S, VP4-M1stopTranslation defect 2C NTPase (L93P) Potentially misfolded protein 3A(L24S) Potentially misfolded protein 3C proteinase (L70P, L102S, C147R)Potentially misfolded protein 3D polymerase (L107P, F246S, F296S)Potentially misfolded protein CRE “l.o.f.” (C13U/A16C/G19A/C22U/Unfolded RNA G25A/A26U/G27C/A31G) Co-dominant Alleles^(b) 2B (F13S,F17S) 3D polymerase (F30S, F191S) 3A (L8S, F83S) Dominant Alleles^(c)Capsid (VP2-S1P, VP2-S243P, Chimeric encapsidation of VP3-L211S,VP1-L118P) wild-type genomes 2A proteinase (L98P, C109R) Defect inintramolecular cleavage yields toxic precursor 3D polymerase (S291P)Chimeric oligomers CRE (G19A/U40C, C22U/U40C) or Arrested RNAreplication or VPg (3B-Y3H) priming complex ^(a)Greater than 80% ofwild-type control. ^(b)Less than or equal to 80% of wild-type controland greater than R2-PvuII control. ^(c)Less than or equal to R2-PvuIIcontrol.

Example 2 Drug-Sensitive Virus Inhibits Growth of Drug-Resistant Virus,where the Drug Target is a Dominant Target

Methods

The methods are described in Example 1.

Results

The Presence of a Drug-Sensitive Virus Inhibits a Drug-Resistant Virus

The trans-acting, highly oligomeric nature of capsid proteins and theobserved dominance of mutant capsid alleles suggested that adrug-resistant virus may inhibit a drug-sensitive virus if a particulardrug targets a capsid protein. Disoxaril (WIN-51711) binds to the“canyon” residues of poliovirus virions, and through stabilization ofthe virion structure, prevents the uncoating of the viral genome afterviral cell entry^(26,27). A mutation known to confer WIN resistance,VP1-I192F, was introduced into a cDNA encoding Sabin-3, the poliovirusserotype known to be most susceptible to the WIN-51711 (FIGS. 7 a,b)²⁸.

To mimic the situation in which a drug-resistant virus would appear in acell infected with wild-type, drug-sensitive virus, co-infections ofwild-type and WIN-resistant polioviruses were performed at a highmultiplicity of infection (MOI) for the wild-type virus and a much lowerMOI for the drug-resistant virus. As can be seen (FIGS. 7 a,b), theoutput of WIN-resistant virus was greatly reduced when grown in thepresence of drug-sensitive virus, to 7% of the yield from a singleinfection. The effect was similar when the single-cycle co-infectionswere performed in the absence or presence of the selective agent. Theobserved dominance of the drug-sensitive genomes may be due to chimericcapsid formation, which rendered WIN-resistant genomes susceptible tothe drug, being partially encapsidated by WIN-sensitive capsid proteins.An alternative explanation, however, was that RNA replication of theWIN-resistant virus was reduced by an unknown mechanism in theco-infection. To test this possibility explicitly, total RNA from allthe infections in FIG. 7 a was subjected to RT-PCR. Differentialdigestion with a restriction enzyme was employed to determine theproportion of WIN-resistant genomes in each infection (FIGS. 7 c,d). Asshown in FIG. 7 e, similar amounts of WIN-resistant viral RNA werepresent in both single and co-infections. The result argues that thereduction in WIN-resistant virus during co-infection was not due to adecrease in RNA replication, but to the formation of chimeric capsidsthat rendered the drug-resistant genome drug-sensitive.

FIGS. 7A-E. Co-infections of drug-sensitive and drug-resistant viruses.Viral infections were performed using either the poliovirus type-3isolate “Fox” strain designated “WIN-S” or a WIN-51711-resistantderivative of the Sabin-3 strain containing a point mutation, VP1-I192F,labeled “WIN-R”. Infections were performed singly or as co-infections,for a single round of virus growth at the indicated MOIs. After virusadsorption, virus growth continued in the absence (a) or presence (b) of2 μg/ml WIN-51711. To measure “total virus”, viral titers weredetermined in the absence of drug; WIN-R virus was measured by addingdrug to virus stock dilutions and agar overlays. Percentages refer tothe relative amounts of WIN-R virus when WIN-R virus from a singleinfection is defined as 100 percent with standard error measurementsindicated by error bars. (c) Schematic of RT-PCR strategy used tomeasure ratio of WIN-S to WIN-R intracellular RNA. Total intracellularRNA was harvested after viral infection of HeLa cells grown in theabsence of WIN at the MOIs indicated, subjected to RT-PCR using primerscommon to both WIN-S and WIN-R RNAs, and digested with a restrictionenzyme, TfiI, to quantify the relative abundances of each RNA species.The asterisk (*) denotes the radiolabeled forward primer; thedifferential mobility of the restriction digested, radiolabeled RT-PCRproduct is used to distinguish WIN-R and WIN-S species in d,e. (d)Standard curve of in vitro transcribed WIN-R and WIN-S RNA. Theindicated RNAs were produced from linearized cDNA templates in vitro andadded to each RT-PCR reaction. The percent of total RT-PCR product thatmigrated as either WIN-S (white bars) or WIN-R (shaded bars) is shownfor each reaction. The relative intensity of each product was quantifiedusing a phosphorimaging plate and ImageQuant software (e) Quantitationof viral intracellular RNA from infected cells. RT-PCR reactions wereperformed as described in c using intracellular RNA harvested from theinfections indicated in a. Each lane is labeled with the infection usedin the RT-PCR reaction. “Mix” refers to a mixing of intracellular RNAsharvested from separate single infections of WIN-R and WIN-S viruses,while “co-infect.” refers to the intracellular RNA harvested from aco-infection of WIN-R and WIN-S viruses. The relative mobilities ofWIN-R and WIN-S digested products are indicated in the left panel. Todetermine the relative abundance of WIN-S and WIN-R RNA species, theindicated bands were quantified using a phosphorimaging plate andImageQuant software. The percent of total signal for WIN-S (white bars)or WIN-R (grey bars) are indicated in the right panel graph.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of identifying a candidate anti-viral agent, the methodcomprising: a) culturing a mammalian cell in vitro in the presence of atest agent, wherein the mammalian cell comprises: i) a parent RNA virus,wherein growth of the parent virus is inhibited by the test agent; andii) a variant of the parent RNA virus, wherein growth of the variant RNAvirus is resistant to the test agent; and b) determining the effect ofparent virus growth on growth of the variant virus during at least onereplicative cycle, wherein, when parent virus growth interferes withvariant virus growth during at least one replicative cycle, the testagent is considered a candidate anti-viral agent.
 2. The method of claim1, wherein, in the presence of the candidate anti-viral agent, parentsuppresses growth of any drug-resistant variant virus for at least onereplicative cycle.
 3. The method of claim 1, wherein the variant RNAvirus genome comprises one or more changes in nucleotide sequencerelative to the nucleotide sequence of the parent RNA virus, wherein theone or more changes in nucleotide sequence result in a change in the RNAand/or an encoded protein that inhibits growth of parent virus in amammalian cell containing both parent virus and variant virus when thecell is cultured in the absence of the test agent.
 4. The method ofclaim 3, wherein the encoded protein is an oligomeric protein.
 5. Themethod of claim 3, wherein the encoded protein is a trans-actingprotein.
 6. The method of claim 1, wherein the parent and the variantRNA viruses are positive-strand RNA viruses.
 7. The method of claim 1,wherein the parent and the variant RNA viruses are negative-strand RNAviruses.
 8. The method of claim 1, further comprising the step ofselecting for the variant of the parent RNA virus.
 9. The method ofclaim 1, wherein the parent virus is a poliovirus, a retrovirus, or ahepatitis C virus.
 10. The method of claim 4, wherein the oligomericprotein is a capsid protein, a membrane-associated protein, or apolymerase.
 11. The method of claim 5, wherein the trans-acting proteinis a protease that cleaves a polyprotein, a protein primer, or an RNAhelicase.